The overall goal of this renewal proposal is to dissect genetic transcription mechanisms in the human dopamine transporter gene (hDAT). The dopamine transporter (DAT) regulates the spatio-temporal domains of dopamine neurotransmission by reuptake and release of dopamine and thus contributes to locomotion, motivation, cognition and attention, working memory, behavioral organization and hormone release. It is well recognized that expression of the DAT gene in the brain is highly circumscribed, varies among individual subjects and can be regulated by endogenous and exogenous factors such as substance uses and stress. Altered DAT expression may contribute to hDAT-associated pathophysiological states such as substance use disorders (SUDs). However, information about how hDAT expression is regulated and how DNA sequence variation influences the regulated expression remains largely sporadic. The hypothesis to be tested in this proposal is that novel transcription factors (TFs) play a major role in regulating the hDAT promoter via disorder- associated cis-acting sites. Our preliminary studies show that TFs may regulate the hDAT promoter in an interactive manner. Therefore, three specific aims of this project are to: 1) demonstrate a RNA-protein complex involved in the promoter regulation; 2) confirm a TF-mediated antagonism between 5? and 3? cis-acting sites; and 3) identify TFs that bind to two functional VNTRs for promoter regulation. The results will add fundamental knowledge on lead sites through which hDAT is regulated by signaling pathways and confers risks for related brain disorders.